The invention concerns a method for the quantitative determination of glycated protein and especially haemoglobin (HbA1c), which is essentially composed of an enzymatic and a chromatographic method step. Subsequently the exact amount of HbA1c is determined by a differential measurement of for example glycated and non-glycated haemoglobin (HbA0). Alternatively the absolute content of glycated and non-glycated proteins can be determined by calibration with suitable standard materials.
There are numerous methods for determining glycated proteins. These can be basically divided into three groups depending on the way in which glycated and non-glycated protein components are separated and quantified (Clin. Chemistry 32 (1986), B64-B70). The first group consists of physicochemical methods based on the utilization of charge differences. These include the HPLC determination with cation exchanger columns such as Diamat, MonoS and PolyCat A (Bisse method) which are the most frequently used methods in clinical chemistry. In the case of haemoglobin for example the quantitative evaluation is carried out by a relative measurement of the HbA1c signal in relation to the total amount of Hb (% HbA1c).
Methods in the second group are those which utilize the different chemical reactivity of glycated and non-glycated protein. These include the thiobarbituric acid method in which for example the glucose bound to haemoglobin is converted into a yellow dye and measured photometrically and also affinity chromatography methods in which complex formation between the vicinal diol groups of the sugar residue and a boric acid group that is bound covalently to a support is used to separate glycated and non-glycated haemoglobin. The separated substance classes are quantified photometrically and the relative amount of glycated haemoglobin is calculated or, in the case of the thiobarbituric acid method quantified, as an absolute determination by calibration with suitable standard materials.
Thirdly, immunological methods may be mentioned. Specific antibodies are used in immunological methods. These recognize for example the structural unit at the N-terminal end of the .beta. chain of the glycated haemoglobin molecule which is typical for HbA1c (e.g. Tina quant.RTM. HbA1c, Boehringer Mannheim). In the immunological methods the absolute content of the respective protein is determined. This necessitates the use of calibrators which have been assigned a target concentration by an independent method. The relative content of for example HbA1c cannot be obtained by a direct measurement.
The known methods are, however, associated with a number of disadvantages. Thus some of the physicochemical methods have a very poor selectivity since the measured signals of glycated protein are overlapped by the non-glycated variants. The shapes of the chromatographic peaks are often asymmetrical and difficult to integrate. The cation exchanger columns that are used are susceptible to small changes in the working conditions and to contamination. Due to the poor selectivity there is a high risk of measuring values that are too high (false positive values).
In the case of chemical methods it is difficult to standardize the procedure and interferences by other components containing sugar can only be avoided with a large amount of effort. It is not possible to differentiate between for example HbA1c and other glycated haemoglobin variants.
The immunological methods are characterized by a very high selectivity towards glycated protein variants. However, the quality of the results depends on the quality of the standard used for calibration. Suitable primary standards in an optimal quality are not at present available, in particular for HbA1c. Information on matrix dependencies cannot be obtained due to a lack of a suitable reference method. In this case false positive values are also frequently obtained (see for example J. Clin. Lab. Anal. 8, (1994), 128-134).